Gene therapy is a method in which a therapeutic gene is delivered to a target organ in vivo so that a new protein is expressed in cells to treat disease. It is not a method of treating the symptoms of disease, but is a method of treating by removing the cause of the disease. Gene therapy may have high selectivity compared to treatment with general drugs and improve the cure rate of disease difficult to control by other therapeutic methods, and thus it can be applied for a long period of time. DNA, a therapeutic gene, is susceptible to hydrolysis by enzymes in vivo and introduced into cells with low efficiency. For this reason, for effective gene therapy, it is required to develop a gene carrier that can safely deliver a therapeutic gene to a target cell to achieve high expression efficiency.
Gene carriers should have low or no toxicity and should be capable to deliver a therapeutic gene to a target cell in a selective and effective manner. Such gene carriers can be largely divided into viral gene carriers and non-viral gene carriers. So far, in clinical trials, viral vectors having high transfection efficiency have been used as gene carriers. However, viral vectors such as retrovirus, adenovirus or adeno-associated virus have problems in that they are prepared by complex processes and pose safety-related concerns, including immunogenicity, infection possibility, proinflammatory potential, and non-specific insertion of DNA and in that the size of DNA capable of being received therein is limited. Due to such problems, the application of such gene carrier to the human body is significantly limited. For this reason, non-viral vectors are receiving attention as an alternative to viral vectors.
Non-viral vectors have advantages in that they can be repeatedly administered with minimal immune responses, can be delivered specifically to a target cell, have excellent storage stability, and are easily produced in large amounts. Examples of such non-viral vectors include cationic liposomes such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), alkylammonium, cationic cholesterol derivatives, gramicidin and the like.
In recent years, cationic polymers among non-viral vectors have received a lot of attention, because they can complexes by ionic bonding with negatively charged DNA. Such cationic polymers include poly-L-lysine (PLL), poly(4-hydroxy-L-proline ester), polyethyleneimine (PEI), poly[α-(4-aminobutyl)-L-glycolic acid], polyamidoamine dendrimers, poly[N,N′-(dimethylamino)ethyl]methacrylate (PDMAEMA) and the like. These cationic polymers can condense DNA to form nanoparticles to thereby protect DNA from enzymatic degradation, and allow DNA to penetrate rapidly into cells and to be released from endosomes. Most non-viral vectors have advantages over viral vectors, including biodegradability, low toxicity, non-immunogenicity, and convenience in use, but have problems, including relatively low transfection efficiency, limited particle size, and the like.
Particularly, most cationic polymers that are used as non-viral vectors show high transfection efficiency in an in vitro environment having low serum concentration, but have problems in that the efficiency of transfection of cationic polymer/gene complexes is significantly reduced by various factors present in serum in an in vivo environment so that the introduction of the gene into cells is not smooth. This is because excessive positive charges occur on the surface of cationic polymer/gene complexes in vivo to cause non-specific interactions with plasma proteins and blood constituents. Thus, in an in vivo environment in which a large amount of serum exists, as opposed to a serum-free medium in vitro or an environment in which serum exists at a very low concentration, the transfection efficiency of cationic polymers is significantly reduced. If these cationic polymers are applied in vivo, they can be agglomerated and accumulated in the lung, liver and spleen and opsonized and removed by the reticuoendothelial system. Thus, the therapeutic application of these cationic polymers can be greatly limited. Polyethyleneimine (PEI) that has been most extensively studied as a non-viral vector has also problems, including low in vivo transfection efficiency, high cytotoxicity, and a low gene expression effect due to low blood compatibility. Accordingly, there is an urgent need to develop a gene carrier which has enhanced transfection efficiency while maintaining the advantages of existing non-viral vectors.
Meanwhile, vitamin B6 (VB6) is a coenzyme that is involved in various cellular metabolisms, including DNA biosynthesis essential for the growth or proliferation of cells. VB6 is taken up by cells through facilitated diffusion via VB6 transporting membrane carrier (VTC) present on the cell membrane. Particularly, because the growth and proliferation of cancer cells actively occur, cancer cells require a large amount of vitamin B6 compared to general adult cells.
Accordingly, the present inventors have made extensive efforts to develop a gene carrier, which has low cytotoxicity, shows high transfection efficiency and can deliver a gene specifically to cancer cells. As a result, the present inventors have developed a vitamin B6-coupled poly(ester amine) gene carrier and have found that the gene carrier shows high transfection efficiency by increasing the accessibility of a complex of a gene and the gene carrier to the cell membrane using vitamin B6 receptors present on the cell membrane, thereby completing the present invention.